1. Assist. Prof. Betül AKCESME05
General Microbiology BIO306
Assist. Prof. Betül AKCESME

2. Nutritions
Elemental and macromolecular composition of a bacterial cell. (a) A microbial periodic table of the elements. With the exception of uranium, which can be metabolized by some prokaryotes, elements in period 7 or beyond in the complete periodic table of the elements are not known to be metabolized.
(b) Contributions of the essential elements to cell dry weight. (c) Relative abundance of macromolecules in a bacterial cell. Data in (b) from Aquat. Microb. Ecol. 10: 15–27 (1996) and in (c) from Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. ASM, Washington, DC (1996).

3. Nutrition 4.1 Metabolism Catabolic reactions (catabolism)
The sum total of all chemical reactions that occur in a cell
Catabolic reactions (catabolism)
Energy-releasing metabolic reactions
Anabolic reactions (anabolism)
Energy-requiring metabolic reactions
Most knowledge of microbial metabolism is based on study of laboratory cultures
Catabolism breaks molecular structures down, releasing energy in the process
anabolism uses energy to build larger molecules from smaller ones.

4. Nutrients Macronutrients Micronutrients
Supply of monomers (or precursors of) required by cells for growth
Macronutrients
Nutrients required in large amounts
Micronutrients
Nutrients required in trace amount

5. Macronutrients Carbon Nitrogen Required by all cells
Typical bacterial cell ~50% carbon (by dry weight)
Major element in all classes of macromolecules (proteins, nucleic acids, lipids, and polysaccharides)
Heterotrophs use organic carbon. use them to make new cell material.
Autotrophs use build their cellular structures from carbon dioxide (CO2)
Nitrogen
Typical bacterial cell ~12% nitrogen   (by dry weight)
Key element in proteins, nucleic acids, and many more cell constituents
hydrogen (H), oxygen (O), carbon (C), nitrogen (N), phosphorus (P), sulfur (S), and selenium (Se).
Amino acids, fatty acids, organic acids, sugars, nitrogen bases, aromatic compounds, and countless other organic compounds can be transported and catabolized
by one or another bacterium.
nitrogen available in nature is in inorganic form as ammonia (NH3), nitrate (NO3 2), or nitrogen gas (N2).

6. Other Macronutrients Phosphorus (P) Sulfur (S) Potassium (K)
Synthesis of nucleic acids and phospholipids
Sulfur (S)
Sulfur-containing amino acids (cysteine and methionine)
Vitamins (e.g., thiamine, biotin, lipoic acid) and coenzyme A
Potassium (K)
Required by enzymes for activity

7. Other Macronutrients Magnesium (Mg) Calcium (Ca) Sodium (Na)
Stabilizes ribosomes, membranes, and nucleic acids
Also required for many enzymes
Calcium (Ca)
Helps stabilize cell walls in microbes
Plays key role in heat stability of endospores
Sodium (Na)
Required by some microbes (e.g., marine microbes)

8. Micronutrients Iron plays a major role in cellular respiration.
Key component of cytochromes and FeS proteins involved in electron transport
Under anoxic conditions, generally ferrous (Fe2+) form; soluble
Under oxic conditions: generally ferric (Fe3+) form; exists as insoluble minerals

10. Micronutrients Growth Factors
Organic compounds required in small amounts by certain organisms
Examples: vitamins, amino acids, purines, pyrimidines
Vitamins
Most commonly required growth factors
Most function as coenyzmes
Although most microorganisms are able to biosynthesize the growth factors they need, some must obtain one or more of them from the environment and thus must be supplied with these compounds when cultured in the laboratory
Coenzymes .which are nonprotein components of enzymes.

11. 4.2 Culture Media Two broad classes
Nutrient solutions used to grow microbes in the laboratory
Most microorganisms in nature have yet to becultured,
Two broad classes
Defined media: precise chemical composition is known
Complex media: composed of digests of chemically undefined substances (e.g., yeast and meat extracts)
The particular carbon source and its concentration depend on the organism to be cultured
Complex media employ digests of microbial, animal or plant products, such as casein (milk protein), beef (beef extract), soybeans (tryptic soy broth), yeast cells (yeast extract), or any of a number of other highly nutritious yet impure substances.

12. Chemoorganotrophs: Escherichia coli and Leuconostoc mesenteroides,
supports growth of the sulfur chemolithotroph Thiobacillus thioparus;

13. Selective Media Differential Media CULTURE MEDIA
Contains compounds that selectively inhibit growth of some microbes but not others
Differential Media
Contains an indicator, usually a dye, that detects particular chemical reactions occurring during growth
CULTURE MEDIA
For successful cultivation of a microbe, it is important to know the nutritional requirements and supply them in proper form and proportions in a culture medium!!!
Differential media are quite useful for distinguishing different species of bacteria and are therefore widely used in clinical diagnostics and systematic
microbiology

14. Pure culture: culture containing only a single kind of microbe
4.3 Laboratory Culture 
Pure culture: culture containing only a single kind of microbe
Contaminants: unwanted organisms in a culture
Cells can be grown in liquid or solid culture media
Solid media are prepared by addition of a gelling agent (agar or gelatin)
When grown on solid media, cells form isolated masses (colonies)
İnoculation-The introduction of a microorganism into a culture medium

15. Produce pigments that cause the colony to be colored
Solid media immobilize cells, allowing them to grow and form visible, isolated masses called colonies
Various shapes and sizes depending on the organism, the culture conditions, the nutrient supply and ect.
Produce pigments that cause the colony to be colored
Contaminated culture !
Figure: Bacterial colonies. Colonies are visible masses of cells formed from the division of one or a few cells and can contain over a billion (10 to 9) individual cells.
(a) Serratia marcescens, grown on MacConkey agar.
(b) Close-up of colonies outlined in part a.
(c) Pseudomonas aeruginosa, grown on trypticase soy agar.
(d) Shigella flexneri, grown on MacConkey agar.
Microbial colonies are of various shapes and sizes depending on the organism, the culture conditions, the nutrient supply, and several
other physiological parameters, and can contain several billion individual cells.

16. Microbes are everywhere
Sterilization of media is critical
Aseptic technique should be followed 

18. Microbiological Methods
Making Media
Pouring Culture Plates
Sterile Technique
Inoculating Plates and Culture Tubes
Use of a Plate Counter to Estimate Microbial Population Densities

19. Culturing Microorganisms
There are two basic culture techniques used in microbiology:
Liquid culture: bacteria, algae, and some fungi can be reared in culture tubes (test tubes) in a liquid medium.
Liquid medium is best when you want to rapidly increase the concentration of the organism or when you want to grow motile cells.

20. Culturing Microorganisms
There are two basic culture techniques used in microbiology:
Culture Plates: Liquid medium is solidified using agar (agarose) and poured as a thin layer in the bottom of a culture dish (also sometimes called petri plate)
Culture plates are used when you want to test (1) antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate individual colonies from environmental samples.

21. Sterile Technique

22. Sterile Technique
When culturing bacteria or other microorganisms, it is important to keep your work area as clean as possible.
This prevents the introduction of other microorganisms from the environment into your culture.
The techniques used to prevent contamination are referred to as sterile techniques.

23. Sterile Technique
Start by washing your down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are:
10% bleach
85% ethanol

24. Sterile Technique (2)
Turn off any forced air heating or air conditioning units that create strong air current in your work area.
A small room or closet that can be closed off is worth the effort to set-up if you will be doing a lot of microbial culturing.
You can install a UV bulb in a fluorescent light fixture to surface sterilize your work bench if you have an enclosed area. Remember to leave the area when you turn on the UV light source!

25. Sterile Technique (3)
All glassware should be cleaned and sterilized before you begin.
All pipettes, spatulas, and test tube (culture) racks should also be sterilized.
You can purchase sterile, disposable culture tubes, petri dishes, and pipettes to minimize the quantity of glassware that you have to sterilize.

26. Sterile Technique (4)
Don’t forget to wash your hands after you finish cleaning and put on a pair of sterile disposable gloves before you begin.
Once your work area is clean, your hands are clean, and your glassware is clean and sterile, don’t contaminate the work area by placing “dirty items” such as pencils, pens, notes, or books in the sterile work area.

27. Media Preparation

28. Microbiological Media
The type of growth medium that you use is a function of the organisms that you want to culture. Use a reference book (there are many) to determine the type of medium that is best suited for your organism of interest.
Common media include Luria Broth (LB), Nutrient Agar, Potato-Dextrose Agar (PDA), Bold’s Basal Medium (BBM)….

29. Luria Broth Liquid Medium 10 g Bacto-Tryptone 5 g Bacto-yeast extract
5 g NaCl
Distilled H2O to 1 l volume
Adjust pH to 7.0
Sterilize for 45 minutes using autoclave or pressure cooker
Plate Medium
10 g Bacto-Tryptone
5 g Bacto-yeast extract
5 g NaCl
Distilled H2O to 1 l volume
20 g agarose
Adjust pH to 7.0
Sterilize for 45 minutes using autoclave or pressure cooker

30. Luria Broth Things to remember:
The volume of media (liquid or plate) should be roughly ½ the volume of the container in which it is placed for sterilization realizing that the liquid expands under increased heat and pressure during the sterilization process.
Estimate plate quantities (how many you need to make) as a function of ml per plate.

31. Assemble all of your chemicals in your work area before you begin.

32. Accurately weigh each of the dry ingredients in your culture media.

33. Add each dry culture medium ingredient to the culture flask.

34. Add distilled (or deionized) water to make the correct volume
Add distilled (or deionized) water to make the correct volume. Heat AND stir (agar will burn if it is not stirred) until all of the ingredients go into solution. When the media boils, it is ready for sterilization.

35. Media Sterilization
There are two reliable methods used to sterilize microbial culture media:
autoclave
pressure cooker
When using an autoclave, use the “wet” setting for sterilizing liquids (flasks, bottles, culture tubes, etc), and use the “dry” setting when sterilizing empty containers, stoppers, etc.

36. Media Sterilization (2)
All liquid media should be sterilized for a minimum for 45 minutes at high temperature and pressure. Autoclaves will cycle automatically, but if you use a pressure cooker, set a timer.
Remember not to tighten the cap or seal on any container; it will explode under high pressure and temperature!
Teachers, please note safety precaution regarding tightening lids on containers prior to sterilization.

37. Sterilize for 45 minutes using the wet cycle (autoclave) or at maximum pressure in a pressure cooker. Remember to cover the top of the flask or jar with aluminum foil to prevent contamination when as the media cools.

38. Plate Pouring Tips Line empty plates along the edge of the work bench.
Open the petri dish lid at about a 30-45° angle to allow the hot liquid to cover the bottom of the dish. The thermal current created by the hot media prevents bacteria and fungal spores from landing in your clean dish.

39. Line your sterile petri plates along the edge of the table
Line your sterile petri plates along the edge of the table. Transfer hot media to a small sterile container and pour ml of the plate media into each petri plate. The petri plate lid should be open slightly, but not completely open as this increases contamination.

40. Plate Pouring Tips
As the plates are poured, move the filled plates to the back of the table until the plates cool and congeal.
Once the plates have cooled and the media is firm, store the plates media side-up (bottom) with the lid securely taped or the plates restacked in the manufacturer’s plastic sleeve.
To increase the shelf-life of the plates, store in a cool, dry environment until they are used (refrigerator).

41. Inoculating Plates and Culture Tubes

42. Inoculation of Culture Plates and Tubes
Clean and surface sterilize your work area as detailed in the section on Sterile Technique.
Use either disposable inoculation loops or a metal loop that can be heat sterilized to inoculate plates, slants, and liquid culture tubes.
If using a metal loop, be sure to cool the loop by touching the sterile cooled liquid media or the sterile culture plate before the placing the loop in your live culture. Failure to cool the loop will kill your active microbial cultures!

43. If gas is unavailable in your lab area, you can modify a standard Bunsen burner to use camp stove propane containers as fuel.

44. Inoculation of Liquid and Solid (Slant) Culture Tubes
Step 1: Remove the culture tube stopper or cap with one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture.

45. Note that the culture tube cap is in the right hand as is the inoculation loop. The sterile culture tube is in student’s left hand. Use your dominant hand to hold the loop. DO NOT put the culture tube cap on the lab bench as this will potentially contaminate your new culture. It is not easy to hold the loop, culture tube, and cap without putting anything down on the bench. We suggest that you have your students practice this technique several times before using actively growing cultures that you want to keep sterile.

46. Inoculation of Liquid and Solid (Slant) Culture Tubes
Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture.
Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.
Step 4: Without setting the loop down, pick-up a sterile fresh culture tube with media with one hand, and remove the cap with the other hand.

47. Inoculation of Liquid and Solid (Slant) Culture Tubes
Step 5: Flame the mouth of the clean culture tube.
Step 6: Place the inoculation loop containing the microbes in the fresh media and swirl the loop in the loop in the media to ensure even dispersal in the media.
Step 7: If using a solid media slant tube, follow steps 1-5 and then zig-zag the inoculation loop across the slanted surface of the solid media in the tube.

48. Note that the culture tube cap is still in the student’s right hand while she holds the liquid culture tube with her left hand. She uses her dominant hand to place the loop in the sterile media.

49. Inoculation of Liquid and Solid (Slant) Culture Tubes
Step 8: Flame the mouth of the newly inoculated culture tube and replace the cap.
Step 9: Place the culture tube in test tube rack.
Step 10: Repeat until all of the sterile tubes have been inoculated. Use a fresh disposable culture loop for each tube or flame the metal loop after each tube has been inoculated.

50. Inoculation of Liquid and Solid (Slant) Culture Tubes
Step 11: Incubate the culture at the recommended temperature (check with your supplier for growth requirements). If using environmental samples, incubation at room temperature will avoid the accidental culture of human pathogens.
Step 12: Dispose of all culture materials in a biohazard bag and sterilize all old cultures before pouring out cultures and washing culture tubes. Disposable culture dishes should be melted in an autoclave or pressure cooker prior to disposal.

51. Safety Note: Use orange biohazard bags to dispose of all solid waste (disposable loops, used gloves, old culture plates) associated with microbial cultures. These bags are engineered to take the heat and pressure of an autoclave without breaking so that contaminated materials are handled safely. Scientific supply houses also sell autoclave tape; this tape changes color or surface pattern when exposed to high heat and pressure. This tape can be used to remind you what has been sterilized and what needs to be sterilized prior to disposal.

52. Inoculating Petri Plates
Step 1:Remove the culture tube stopper or cap with one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture.
Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture.
Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.

53. Inoculating Petri Plates
Step 4: Holding the petri dish lid at an 30-45° angle, work the inoculating loop from the outside of the plate toward the center in a zig-zag pattern that covers approximately 25% of the plate surface (think pie or pizza slice!).

55. Inoculating Petri Plates
Step 5: Turn the petri plate 90° to the right, dragging the inoculation loop through the last section of the plate, moving from the outside to the inside in a zig-zag motion.
Step 6: Repeat this process twice more until the entire plate surface is covered.
NOTE: If you are trying to isolate individual colonies, each turn of the dish will give you fewer microbes so that you can distinguish individual colonies.

56. If you streak your plates correctly, the pattern should look like this on your plate.

57. Use of a Plate Counter for Estimating Microbial Populations

58. Serial Dilution of Environmental Samples or Commercial Cultures
Serial dilution techniques should be used in the estimation of microbial population sizes.
Serial dilution involves the use of a known amount (in ml or μl) in a known volume of liquid media.
A one in ten dilution is made in a new liquid culture tube, and this process is usually repeated several times. The resulting cultures are dilutions of 1/10, 1/100, 1/1000, 1/10,000, for example, of the original sample.
These cultures are plated on petri plates and incubated at the recommended temperature.

59. Estimating Microbial Population Size
After the inoculated plates are incubated for the appropriate time period, the number of colonies per plate are counted.
Population estimates are obtained by multiplying the dilution factor by the number of colonies per plate. The resulting number is a rate (function) of the initial weight or initial volume used from the environmental sample or culture (per gram soil, per ml or μl of culture).

60. Counting Plates
If a commercial plate counter is not available, you can Xerox 1 mm square graph paper and use it as a grid for colony counting. You would need to estimate the total surface area (in mm2) by counting the number of squares in a dish.
If using a commercial plate counter, touch each colony on the plate with the pen, and the cumulative number of colonies will appear on the display.

63. Summary
Different media are used to culture microorganisms, be certain that you are using the appropriate media for your organism.
Always use sterile technique to prevent contamination.
Choose the type of media (liquid or plate) appropriate for your investigation or application.
Sterile liquid culture tubes and media plates can be prepared in advance and stored in the refrigerator for later use (2 weeks for liquid culture tubes, 2 months for media plates).

64. Summary
Liquid culture tubes, solid slant tubes, and petri plates can be used to culture microbes.
Media and lab materials should be sterilized prior to use; an autoclave or a pressure cooker can be used in the sterilization process.
Serial dilution and plate count techniques are used to estimate microbial populations from environmental or commercial cultures.